| 谭小艳,马耀华,郝兆祥.4种改良CTAB法提取石榴成熟叶片DNA的比较研究[J].中国南方果树,2021,50(3): |
| 4种改良CTAB法提取石榴成熟叶片DNA的比较研究 |
| Comparison and Application of Four Improved CTAB Extraction Methods from Mature Leaves of Pomegranate |
| 投稿时间：2020-06-01 修订日期：2020-12-22 |
| DOI： |
| 中文关键词: 石榴 成熟叶片 改良CTAB法 基因组DNA 提取 |
| 英文关键词:Pomegranate (Punica granatum L.) Mature leaves The improved CTAB method Genomic DNA Extraction |
| 基金项目:山东省科技发展计划项目(2011GGA04012)；国家林业公益性行业科研专项资助项目(201204402)；国家林业局林木种苗工程资助项目([2012]888号)；枣庄市科学技术发展计划项目(201233-2)。 |
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| 中文摘要: |
| 【目的】研究从成熟石榴叶片中提取高质量基因组DNA的方法。【方法】根据成熟石榴叶片组织中富含多酚、多糖的特点,以峄城中国石榴种质资源圃20种石榴成熟叶片为材料,分别采用改良CTAB法(Ⅰ、Ⅱ、Ⅲ、Ⅳ)4种方法提取成熟石榴基因组DNA,通过琼脂糖凝胶、紫外分光光度计分析、ISSR扩增等方法检测所提取DNA的质量。【结果】改良的CTAB Ⅳ法能从各个石榴品种的成熟叶片中提取出基因组DNA,琼脂糖电泳胶孔干净,无拖带,且DNA的纯度和完整性都较好,OD260 nm/OD280 nm的比值均在1.8~2.0之间,无降解现象,其质量、产量都高于其他改良CTAB法。经ISSR- PCR扩增结果表明,此方法提取的基因组总DNA完全适于ISSR分析。其主要的步骤是：在石榴叶片的研磨中添加Vc、PVP等抗氧化物质,加入CTAB提取液后65 ℃水浴30 min后冷却至室温,离心条件为15℃,10 000 r/min,10 min,DNA粗提液用氯仿抽提3次,可获得高质量的DNA。【结论】改良的CTAB Ⅳ法能有效地从石榴成熟叶片中提取到高质量的基因组DNA。 |
| 英文摘要: |
| [Objective] To develop an efficient and stable method for extracting high-quality genomic DNA from mature leaves of pomegranate (Punica granatum L.). [Method] The mature leaves of pomegranate are rich in polyphenols, polysaccharides, 20 pomegranate cultivars leaves from China germplasm nursery at Yicheng of Zaozhuang were used as studying materials. The genomic DNA was extracted using the improved CTAB method (Ⅰ, Ⅱ, Ⅲ, Ⅳ), and the purity and quantity of each DNA sample obtained was evaluated by agarose gel electrophoresis, UV scanning and ISSR polymorphic analysis. [Results] Each of the genomic DNA extracted from mature leaves of 20 pomegranate cultivars by this method was pure, integral, and the sample hole was clean and no pulling, the value of OD260 nm/OD280 nm was between 1.8 to 2.0, no degradation phenomena. And its quality and yield was high than that extracted by other improved CTAB methods. The ISSR- PCR amplification results showed that this method of extracting total genomic DNA meet the analysis requirements. The main steps are: add antioxidants: Vc and PVP in the milling process of the leaves, 65 ℃ water bath for 30 min after add the CTAB buffer, cooling to room temperature, centrifugation conditions are 15 ℃, 10 000 r/min and10 min. The crude DNA was extracted 3 times with chloroform, can high-quality DNA is obtained. [Conclusion] The improved CTABⅣ method could be used for extracting high-quality genomic DNA from matured pomegranate leaves effectively. |
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